Study On Secondary Metabolites Of Three Plant-derived Endophytic Fungi

Plant-derived endophytic fungi possess distinctive and complex metabolic pathways, thus they can produce secondary metabolites with variety structures, novel skeletons and diversity biological activities. In this paper, the secondary metabolites of three endophytic fungi from two plants(two strains from Orchidantha.chinensisi and one strain from Crocus Sativus L.) were systematically studied including isolation and screening active endophytic fungi from O.chinensisi, purification and structure identification of the secondary metabolites of endophytic fungi from O. chinensisi and C. Sativus L., and preliminary bioactivities evaluation of some pure compounds.In the first chapter, the definition and diversity of endophytic fungi, and the research progress of the bioactive compounds from plant-derived endophytic fungi in recent years were summarized. Finally, the study process of endangered medicinal plant O. chinensisi and valuable medicinal plant C. Sativus L. were described,respectly. The whole herb of O. chinensis has been used as a traditional folk antitussive and antipyretic medicine, and had moistening lung and relieving cough efficacy. However, due to the destruction of environment and the decline in their ability to reproducec it has been listed as an endangered plant. The stigma of saffron possess of invigorating the circulation of blood, curing anxiety, depression and insomnia. Saffron has been known as “gold plant” because the low yield of ingredients contained in the stigmas.The second chapter described forty-two strains endophytic fungi were isolated from leaf, petiole, root and rhizome of O.chinensisi. The endophytic fungi were screened using the chemical and biological activity screening phases. In this process of chemical screening phase, TLC was used. And the chemical diversification were the objectives for atudy. In this process of biological activity screening phase, it was focused on antibacterial(Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coil and so on) activities. By the comprehensive analysis of chemical andbiological results, two target strains(OC42 and OC4) have been selected for further study.The third chapter reported that the endophytic fungus OC42 was fermented 100 L by shaking cultivation. The culture broth was concentrated, then extracted with ethyl acetate and concentrated under reduced pressure to gain crude extract; the mycelium was immersed with methanol and extracted with ethyl acetate and concentrated in vacu to afford extract. Two extracts were combined and investigated their ingredients by silica gel column chromatography, Sephadex LH-20 column chromatography and recrystallization. Eleven compounds were obtained and structures of ten compounds were unequivocally elucidated on the basis of NMR, MS and IR spectrum, which are(9S, 2S, 13S)-trihydroxy-10E-octadecenoic acid(OC42-1),p-hydroxybenzoic acid(OC42-2), N-(2-amino-2-oxoethyl)benzamide(OC42-3),thymine(OC42-4), erytro-9, 10-dihydroxistearic acid(OC42-5),(22E, 24S)-5α,8α-epidioxy-24-methyl-cholesta-6, 9(11), 22-trien-3β-ol(OC42-6),(22E, 24S)-5α,8α-epidioxy-24-methyl-cholesta-6, 22-dien-3β-ol(OC42-7), ergosterol(OC42-8),(22E, 24R)-cerevisterol(OC42-9) and 2, 3-dihydroxypropyl heptadecanoate(OC42-10). Compound(OC42-3) was determined to be a novel natural product.The forth chapter reported the cultivation of the endophytic fungus OC4 by solid state fermentation, then the rice culture was immersed with methanol and extracted with ethyl acetate and concentrated in vacu to afford extract. Three compounds were afforded by repeated column chromatography on silica gel, Sephadex LH-20 and recrystallization. By means of modern spectroscopic methods, their structures were identified as(2S, 5R)-2-ethyl-5-methylhexanedioic acid(OC4-1),(22E,24R)-cerevisterol(OC4-2) and(22E, 24S)-5α, 8α-epidioxy-24-methyl-cholesta-6,22-dien-3β-ol(OC4-3), respectively. Among them, compound(OC4-1) was a new compounds. Then the antibacterial and antioxidant activities of compound(OC4-1)were investigated and the compound displayed strong antioxidant activity with IC50 value of 1.08 mg m L-1 on superoxide anion racdical.The fifth chapter reported the investigation of secondary metabolites of endophytic fungus CSL-13 from C. Sativus L. Our previous work had found theendophytic fungus CSL-13 from C. Sativus L. performed potent antibacterial activity and thus cultivated the endophytic fungus CSL-13 by solid state fermentation, then the rice culture was was immersed with methanol and extracted with ethyl acetate and concentrated in vacu to afford extract. Three compounds were obtained by repeated column chromatography on silica gel, Sephadex LH-20 and recrystallization. By means of modern spectroscopic methods, their structures were assigned as beauvericin(CSL13-1),(22E, 24S)-5α, 8α-epidioxy-24-methyl-cholesta-6,22-dien-3β-ol(CSL13-2) and Prolipyrone C(CSL13-3), respectively. The biosynthetic pathway of CSL13-1 was briefly discussed in the chapter.